We are requesting a Flow Cytometer since Flow Cytometry is one of the major techniques emphasized in the newly established Center for Fluorescence Research in Biomedical Sciences. This instrument will be used in four separate, yet integrated projects: (a) New fluorescent probes will be designed, synthesized and tested by flow cytometry. The proposed new probes include: long wavelength pH indicators, indicators of membrane potential, cell surface markers, and calcium indicators; a long wavelength probe of calmodulin which is sensitive to calcium binding; and a range of fluorescent substrates for specific lysosomal enzymes; (b) the temporal sequence of possible transducing signals such as pH, and free Ca++ will be analyzed during the stimulation of macrophases with the chemotactic factor C5a; (C) some of the biochemical and biophysical consequences of receptor mediated endocytosis will be investigated; and d) bulk injection methods will be optimized for loading cells and for selecting subpopulations of these cells for further analyses by flow and quantitative fluorescence microscopy.